Self-splicing RNAs are capable of catalyzing excision of their own introns in the absence of other protein or RNA factors. Self-splicing RNAs are classified into one of two groups, group I or group II, dependent on the reaction mechanism used to excise the intron. Group II introns excise themselves using a mechanism of action similar to that used in nuclear pre-mRNA splicing. In this mechanism, the 5′ splice site is cleaved by attack from an adenosine nucleotide within the intron. The result is a lariat-like intermediate which is subsequently excised.
Group II introns encode ribonucleoprotein (RNP) particles, referred to hereinafter as “nucleotide integrases”, that comprise an excised group II intron RNA and a group II intron-encoded protein which is bound to the excised group II intron RNA. Nucleotide integrases are molecular complexes capable of cleaving double-stranded DNA substrates at specific recognition sites and inserting nucleic acid molecules into the DNA substrate at the cleaved recognition site. Each nucleotide integrase cleaves substrate DNA and inserts nucleic acid molecules at specific recognition sites in the substrate DNA.
Methods of using nucleotide integrases are described in U.S. Pat. No. 5,698,421 and U.S. Pat. No. 6,027,895, both of which are specifically incorporated herein by reference. The process of cleaving the DNA substrate and inserting nucleic acid molecules involves base pairing of the group II intron RNA of the nucleotide integrase to a specific region of the DNA substrate. Additional interactions occur between the intron-encoded protein and regions in the DNA substrate flanking the recognition site. In general, the method comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two sequences, EBS1 and EBS2, that are capable of hybridizing with two intron RNA-binding sequences, IBS1 and IBS2, on the top strand of the DNA substrate, and a group II intron-encoded protein that binds to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate for a time and at a temperature sufficient to permit the nucleotide integrase to cleave both strands of the DNA substrate and to insert the group II intron RNA into the cleavage site of the top strand. The first sequence element of the recognition site is upstream of the putative cleavage site, the IBS 1 sequence and the IBS 2 sequence. The first sequence element comprises from about 10 to about 12 pairs of nucleotides. The second sequence element of the recognition site is downstream of the putative cleavage site and comprises from about 10 to about 12 nucleotides.
As denoted herein, nucleotides that are located upstream of the cleavage site have a (−) position relative to the cleavage site, and nucleotides that are located downstream of the cleavage site have a (+) position relative to the cleavage site. Thus, in the above-described method, the cleavage site is located between nucleotides −1 and +1 on the top strand of the double-stranded DNA substrate. The IBS1 sequence and the IBS2 sequence lie in a region of the recognition site which extends from about position −1 to about position −14 relative to the cleavage site.
EBS1 is located in domain I of the group II intron RNA and comprises from about 5 to 7 nucleotides that are capable of hybridizing to the nucleotides of the IBS1 sequence of the substrate. EBS2 is located in domain I of the group II intron RNA upstream of EBS1 and comprises from about 5 to 7 nucleotides that are capable of hybridizing to the nucleotides of IBS2 sequence of the substrate. In order to cleave the substrate efficiently, it is referred that the nucleotide or sequence δ, which immediately precedes the first nucleotide of EBS1 of the group II intron RNA, be complementary to the nucleotides at +1 in the top strand of the substrate.
Examples of nucleotide integrases which may be used to catalyze the cleavage of double-stranded DNA molecules are the aI2 nucleotide integrase, the aI1 nucleotide integrase, and the ltrA nucleotide integrase. The aI2 integrase is an isolated RNP particle that comprises a wild-type or modified group II intron RNA of the second intron of the S. cerevisiae mitochondrial COX1 gene, hereinafter referred to as the “aI2 intron” RNA, bound to a wild-type or modified aI2 intron encoded-protein. EBS1 of the aI2 intron RNA comprises 6 nucleotides and is located at position 2985-2990 of the wild-type sequence. EBS1 of the wild-type aI2 intron RNA has the sequence 5′-AGAAGA. EBS2 of the aI2 intron RNA comprises 6 nucleotides and is located at positions 2935-2940. EBS2 of the wild-type aI2 intron RNA has the sequence 5′-UCAUUA.
The aI1 nucleotide integrase is an isolated RNP particle that comprises an excised, wild-type or modified excised group II intron RNA of the first intron of the S. cerevisiae mitochondrial COX1 gene, hereinafter referred to as the “aI1 intron” RNA, and a wild-type or modified aI1 intron-encoded protein. EBS1 of the aI1 intron RNA comprises 6 to 7 nucleotides and is located at position 426-431. EBS1 of the wild-type aI1 intron RNA has the sequence 5′-CGUUGA. EBS2 of the aI1 intron RNA comprises 5 to 6 nucleotides and is located at positions 376-381. EBS2 of the wild-type aI1 intron RNA and has the sequence 5′-ACAAUU.
The ltrA nucleotide integrase comprises an excised, wild-type or modified excised group L1.LtrB group II intron RNA of the Lactococcus lactis ltrB gene, hereinafter referred to as the “L1.ltrB intron” RNA, and a wild-type or modified L1.ltrB intron-encoded protein, hereinafter referred to as the ltrA protein. The sequence of the L1.ltrB intron is shown in FIG. 7. The EBS1 of the L1.ltrB intron RNA comprises 7 nucleotides and is located at positions 457 to 463. The EBS1 sequence of the wild-type L1.ltrB intron RNA has the sequence 5′-GUUGUGG. The EBS2 of the L1.ltrB intron RNA comprises 6 nucleotides and is located at positions 401 to and including 406. The EBS2 sequence of the wild-type L1.ltrB intron RNA has the Sequence 5′AUGUGU.
In recent years, methods have been developed for preparing nucleotide integrases whose excised group II RNAs have a wild-type sequence and nucleotide integrases whose excised group II RNAs have a modified sequence. Such methods are described in U.S. Pat. No. 5,804,418, which is specifically incorporated herein by reference. The modified nucleotide integrases can catalyze the cleavage of DNA substrates and the insertion of nucleic acid molecules at new recognition sites in the DNA substrate. Because the recognition site of the DNA substrate is recognized, in part, through base pairing with the excised group II intron RNA of the nucleotide integrase, it is possible to control the site of nucleic acid insertion within the DNA substrate. This is done by modifying the EBS 1 sequence, the EBS 2 sequence or the δ sequence, or combinations thereof. Such modified group II introns produce nucleotide integrases that can cleave DNA substrates and insert nucleic acid molecules at new recognition sites in the genome.
Modified nucleotide integrases are useful analytical tools for determining the presence and location of a particular target sequence in a DNA substrate. Modified nucleotide integrases are also useful tools for rendering certain genes within DNA substrates nonfunctional. Modified nucleotide integrases are also useful tools for inserting a nucleic acid into the cleavage site, thus changing the characteristics of the cellular DNA and RNA and protein molecules encoded by the cellular DNA.
Accordingly, analytical systems and methods which can be used to determine, first, whether or not a modified nucleotide integrase functions to cleave substrate DNA and insert nucleic acid molecules and, second, to analyze the recognition site where this occurs are desirable.